Summary of Work: There are more than 36 million people infected by the HIV virus worldwide where 5.3 million new infections occurred during 2000. Although antiviral therapy can extend the life of individuals, the death toll continues to rise: 2.6 million people, the highest number since the epidemic began, died from AIDS this year. Current antiviral nucleoside analog therapy against HIV results in compromised mitochondrial function due to selective inhibition of the mitochondrial DNA polymerase. As much as 40% of patients undergoing AZT treatment develop a mitochondrial dysfunctional disease known as red ragged fiber disease. The mode and effect of antiviral nucleotide analogs, by AZT, ddI, 3TC, D4T and others on the inhibition and fidelity of the mitochondrial DNA polymerase and mitochondrial DNA replication are poorly understood. What structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns is poorly understood. We previously evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase gamma (pol gamma) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. The structural elements responsible for this inhibition and the amino acids that interact with these analogs are unknown. In efforts to understand why pol gamma is so sensitive to antiviral nucleotide analogs, we have changed three key amino acids believed to make contacts with the incoming nucleotide. These changes were based on the X-ray crystallographic structure of T7 DNA polymerase and E. coli DNA polymerase I. We changed Tyr951 to Phe and Ala, Tyr955 to Phe and Ala, and Glu895 to Ala. These overproduced, purified mutant gamma polymerases are being screened for their sensitivities to the antiviral nucleotide analogs AZT-TP, 3TC-TP, D4T-TP, ddC-TP, and carbovir and compared with wild-type pol gamma. Additionally, steady state kinetic analysis and DNA binding properties are being determined for these mutant pol gamma proteins. Incorporation of antiviral nucleotides is being assayed using oligonucleotide substrates as previously described.